Poly(ethyleneoxide)-monomethylether (MeO-PEO) (Mn = 350, 500, 750, 2,000, and 5,000), 2-[2-(2-methoxy)ethoxy]acetic acid (MeO-PEO-3-CO2H), 3-mercaptopropanoic acid, 2-aminoethanethiol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4,4′-azobis(4-cyanovaleric acid) (ACVA), 3-aminopropyltriethoxysilane (APTES), ethanoyl chloride, butanoyl chloride, octanoyl chloride, 2-ethylhexanoyl chloride, decanoyl chloride, oleoyl chloride, myristoyl chloride, phenolphthalein (A.C.S. reagent), hydrogen peroxide, triethylamine, toluene, 1,1,2-trichlorotrifluoroethane (A.C.S. reagent), tetrahydrofuran (THF), dansyl chloride, and sodium carbonate were purchased from Aldrich Chemical Company (Gillingham, United Kingdom). Perfluorobutanoyl chloride and perfluorooctanoyl chloride were purchased from Lancaster Synthesis (Lancaster, United Kingdom). Methanol (high-pressure liquid chromatography [HPLC] grade), 2-propanol (HPLC grade), acetone (AR grade), diethyl ether (HPLC grade, peroxide-free), sodium hydrogen carbonate, sodium hydroxide, potassium dichromate, sulfuric acid, and p-toluene sulfonoyl chloride were purchased from Fisher Scientific (Loughborough, United Kingdom). Azobis(isobutyronitrile) (AIBN) was purchased from Acros Organics (Loughborough, United Kingdom) and recrystallized from methanol prior to use. 2-Mercaptoethanol, ethanol (AR grade), and chloroform (AR grade) were purchased from BDH (Poole, United Kingdom). Sulfo-SDTB was purchased from Pierce (Rockford, Ill.). N-Methylacrylamide was purchased from Monomer-Polymer Laboratories (Trevose, Pa.). Krytox acid terminal perfluoropolyether was obtained from DuPont (Deepwater, N.J.). Cytochrome c (from bovine heart) and bovine serum albumin (BSA) were obtained from Sigma (Poole, United Kingdom). d-[U-14C]glucose and 3H-acetic anhydride were obtained from Amersham Life Sciences (Amersham, United Kingdom). Unless otherwise stated, all chemicals were used as received.
Growth and maintenance of microorganisms.
Bacterial cultures were maintained on Tryptone Soya Agar (Oxoid) at 4°C. Stock cultures were grown statically overnight at 37°C in coryneform broth (containing [in grams per liter] tryptone, 10; yeast extract, 5; sodium chloride, 5; and glucose, 5; pH 7.2) for L. monocytogenes C200 type 2 and in yeast-dextrose broth (containing [in grams per liter] peptone, 10; beef extract, 8, sodium chloride, 5; glucose, 5; and yeast extract, 3; pH 6.8) for S. typhimurium LT2 50185, S. aureus NCDO 949, and E. coli NCFB 1989. Aliquots (100 ml) of stock culture in sterile Eppendorf tubes were drop-frozen in liquid nitrogen and stored at −70°C prior to use. Prior to the first adhesion experiments, growth curves were established for the bacteria by correlating plate counts and optical density measurements. Bacteria plated from the stationary phase showed no significant loss in viability after 24 h, as determined by plate counts. Cell viability after the adsorption experiments was qualitatively confirmed by using confocal microscopy, which clearly showed the formation of bacterial colonies on the grafted surfaces and plate counts, by using polymer beads rather than films but with the same surface functionality.
Preparation and characterization of silylated glass surfaces.
Glass coverslips (13-mm diameter; Chance Propper, Ltd., Smethwick, United Kingdom) were hydrolyzed by immersion in sodium hydroxide (aqueous, 5 M) for 1 h and washed thoroughly with deionized water. They were then soaked in fresh piranha solution (70% sulfuric acid, 30% hydrogen peroxide) for 1 h, washed with water, and dried in air. The glass surfaces were prepared immediately prior to silylation with APTES by drying them in a hot oven for 30 min. The coverslips were then immersed in a solution containing 95% acetic acid (1 mM) in methanol, 4% water, and 1% APTES for 30 min at room temperature with occasional gentle shaking. The resultant amine-functional surfaces were finally washed with methanol (three times with 50 ml) and cured in a hot oven for 30 min.
The density of amine groups on APTES-treated glass coverslips was determined by titration with dansyl chloride in chloroform (10 ml, 0.3 mg · ml−1). The reaction was carried out in the dark at room temperature for 2 h. The coverslips were then washed twice with chloroform (5 ml) and three times with ethanol (5 ml) and dried in air, and the fluorescence spectra were recorded. For UV spectroscopy, three coverslips were placed in a quartz cuvette to enable sufficient peak absorptions to be recorded.
The concentration of amine groups on APTES-treated glass beads (prepared as described above by using 100-μm-diameter silica beads) was determined as follows. Sulfo-SDTB (3.2 mg) in N,N1-dimethyl-formamide (2 ml) was added to sodium carbonate buffer (8 ml, 50 mM) to make a standard solution. Aliquots (~3 ml) were added to APTES-treated glass beads (0.5 g) and allowed to stand at room temperature for 1 h. The beads were then washed thrice with methanol (5 ml), twice with water (5 ml), and another three times with methanol (5 ml) prior to the addition of 1:1 methanol-perchloric acid (5 ml). The amine group concentration was determined by measuring the absorption intensity of the liberated dimethoxytrityl cation at 498 nm.
Synthesis of reagents. (i) MeO-PEO-CO2H.
Synthesis was carried out as described previously (13
). The protocol described below is for MeO-PEO-5000, but a similar procedure was used for other PEO oligomers. High-molecular-weight MeO-PEO-OH (5.0 g, 1 mmol) was dissolved in anhydrous DMF (100 ml) with 1.1 eQ of succinic anhydride (0.1 g) in a dry, three-necked 250-ml round-bottomed flask and heated to 100°C under an atmosphere of dry nitrogen overnight. Upon cooling the mixture was concentrated under reduced pressure, and the polymer was purified by precipitation into cold ether (three times, 250 ml), followed by drying in a vacuum oven at 40°C overnight.
(ii) MeO-PEO acyl chlorides.
High-molecular-weight carboxyl-terminated MeO-PEO (5.1 g, 1 mmol) was dissolved in dry toluene (100 ml) in a 250-ml three-necked round-bottomed flask. Oxalyl chloride (0.375 g, 3 mmol) was added, and the mixture was brought to reflux under an atmosphere of dry nitrogen and stirred at reflux overnight. Upon cooling the solvent and excess oxalyl chloride were removed under reduced pressure, and the resultant MeO-PEO-acyl chloride was used immediately. The acyl chloride of 2-[2-(2-methoxy)ethoxy]acetic acid (MeO-PEO-3-COCl) was synthesized in the same way.
(iii) Krytox acyl chloride.
Krytox (5.0 g) was dissolved in 1,1,2-trichlorotrifluoroethane (100 ml) in a 250-ml three-necked flask. Oxalyl chloride (3 ml) was added, and the mixture was brought to reflux under an atmosphere of dry nitrogen and stirred at reflux overnight. Upon cooling the solvent and excess oxalyl chloride were removed under reduced pressure, and the resultant acid chloride was used immediately.
(iv) Poly(N-methylacrylamide) polymers.
In a thick-walled tube, monomer (N-methylacrylamide, 10 g) was dissolved in 2-propanol (40 ml) with chain transfer agent (0.344 mmol) and initiator (AIBN or ACVA [see below], 2.83 mmol). This mixture was degassed by freeze-thaw cycles under vacuum at least three times. The tubes were then placed in a thermostatted oil bath at 65°C for 24 h. After it cooled to room temperature, the mixture was concentrated under reduced pressure, and the residue was added to diethyl ether (250 ml) to precipitate the polymer. This was filtered and the residue was redissolved in THF and reprecipitated into diethyl ether (three times, 250 ml) to leave the purified polymer as a colorless precipitate which was then dried in vacuo at 20°C overnight.
The molecular weight and functionality of the poly(N-methylacrylamide) polymers were controlled with the use of functional initiators and suitable chain transfer agents. Carboxyl-terminated polymers were obtained with the use of 3-mercaptopropanoic acid as a chain transfer agent and ACVA as the free radical initiator. The molecular weight of carboxyl-terminated poly(N-methylacrylamide) (~100 mg) was determined by titration of dissolved polymer in deionized water (50 ml) with freshly prepared sodium hydroxide solution (10 mM). The endpoint was either determined potentiometrically or by using phenolphthalein solution as an indicator. Amine- and hydroxy-terminated polymers were obtained by using AIBN as the free radical initiator and 2-aminoethanethiol or 2-mercaptoethanol as the chain transfer agent.
Chemical modification of surfaces. (i) Treatment with carboxyl-terminated poly(N-methylacrylamide).
Silylated coverslips were placed in dilute hydrochloric acid (pH 6) and cooled to 0°C. Carboxyl-terminated poly(N-methylacrylamide) (1.0 g) was added to the buffer solution, followed by EDC (three times, 200 mg) every 30 min with gentle stirring. After the addition was complete, the surfaces were left at 0°C for a further 72 h. The surfaces were then rinsed repeatedly with deionized water (five times, 250 ml) and dried in air.
(ii) Treatment with acid chlorides.
Glass coverslips were placed in dry chloroform (40 ml) and triethylamine (10 ml); to this the required acid chloride (5 ml) was added, and the mixture was left at room temperature under nitrogen with gentle stirring for 2 h. The coverslips were then washed with chloroform (four times, 50 ml) and dried in air.
(iii) Treatment with Krytox acid chloride.
Glass coverslips were placed in 1,1,2-trichlorotrifluoroethane (10 ml) and triethylamine (3 ml) containing Krytox acid chloride (3 g) and left at room temperature with gentle stirring for 1 h; the coverslips were then washed in a Soxhlet apparatus with 1,1,2-trichlorotrifluoroethane overnight and dried in air.
To remove possible contamination remaining after grafting, the substrates were cleaned by sequential washing with ethanol (three times, 100 ml) and distilled water (three times, 100 ml), followed by rinsing with water and drying in dust-free air.
Assessment of bacterial attachment to synthetic surfaces.
To carry out radioactive labelling of microorganisms, modified coryneform broth (6 ml, containing a growth-limiting concentration of glucose [1 g · liter−1]) for cultures of L. monocytogenes or modified yeast-dextrose broth (6 ml, containing glucose [1 g · liter−1]) for cultures of S. typhimurium, S. aureus, or E. coli were inoculated with 50 μl of thawed stock culture. Aliquots of d-[U-14C]glucose were added to a final concentration of 20 μCi · ml−1 from a 1-mCi stock solution (230 to 370 mCi · ml−1; a sterilized, aqueous solution containing 3% ethanol). The cultures were incubated, statically, at 37°C for 24 h.
Labelled bacterial cultures (6 ml) were transferred to Eppendorf tubes and centrifuged (3 min, 13,000 rpm). The cells were washed twice in sterile MOPS (morpholine propane sulfonic acid; 50 mM, pH 7.0, 6 ml) and finally resuspended in MOPS (6 ml). Aliquots (200 μl) of the cell suspension were transferred to sterile MOPS (10 ml) in 15-ml capped bottles. Modified glass coverslips, stored in absolute ethanol prior to use, were aseptically transferred to the bacterial suspensions after evaporation of the ethanol. The bottles were placed in an oven (Techne Hybridiser HB-1D) and incubated, with gentle shaking, for 24 h, unless otherwise stated. The glass coverslips were rinsed in sterile MOPS (50 mM, pH 7.0, 10 ml) and transferred to scintillation vials containing Packard Insta-Gel Plus scintillant cocktail (5 ml; Canberra Packard Ltd., Pangbourne, United Kingdom). Counting was performed on a Packard 2200CA Tri-carb liquid scintillation analyzer.
Determination of absolute cell counts.
Aliquots (200 μl) of unlabelled bacterial culture were enumerated via a serial dilution method with Tryptone Soya Agar and incubation at 37°C for 24 h. The numbers of viable cells determined this way were compared with the scintillation counts from equivalent aliquots (200 μl) of radiolabelled bacteria. In addition, the scintillation readings were correlated with total bacterial numbers obtained via optical density measurements and microscopy.
Adsorption of proteins to synthetic surfaces.
The method to prepare radioactively labelled proteins was adapted from that used by Freeman and Parish to label heparan (12
). Cytochrome c
or BSA (20 mg in each case) was dissolved in NaHCO3
(aqueous 0.5 M, 1 ml) containing 10% (vol/vol) methanol in a sealed 15-ml reaction vial and cooled to 0°C in an ice bath. 3
H-acetic anhydride (0.1 ml, 10 mCi; 500 mCi · mmol−1
) in toluene was added, and the mixture was stirred for 3 h at 0°C. The mixture was acidified to pH 7.0 with acetic acid and allowed to warm to room temperature, and the toluene was removed under a stream of nitrogen. The solution was desalted by using a PD-10 column (Pharmacia Biotech, St. Albans, United Kingdom) equilibrated and developed with aqueous ethanol (10% [vol/vol]). Column fractions containing radioactive material appearing ahead of the 3
H-acetate peak were pooled, sodium azide was added (0.1% [wt/vol]), and the solution was finally stored at 4°C. Protein concentration was determined by using the Lowry method for BSA and from a spectrophotometric calibration curve (408 nm) for cytochrome c
To determine the protein adsorption to surfaces, radiolabelled BSA (typically 37.5 μg) or cytochrome c (42.5 μg) was added to MOPS (50 mM, pH 7.0, 10 ml) in 15-ml screw-capped bottles. Derivatized glass coverslips, prepared and stored as described above, were transferred to the protein solutions. The bottles were placed in an oven (Techne Hybridiser HB-1D) and incubated, with gentle shaking, for 1 h (unless otherwise stated) at 37°C. At indicated time intervals the coverslips were rinsed twice in MOPS (50 mM, pH 7.0, 10 ml) and transferred to 5-ml scintillation vials. Counting was performed on a Packard 2200CA Tri-carb liquid scintillation analyzer as described above.
Pretreatment of substrates with BSA and cell exudate.
Native BSA (37.5 μg) was added to MOPS (50 mM, pH 7.0, 10 ml) in 15-ml screw-capped bottles. Glass coverslips (13-mm in diameter), previously stored in absolute ethanol, were transferred to the protein solutions. The bottles were incubated, with gentle shaking, for 1 h (unless otherwise stated) at 37°C. The coverslips were then rinsed in sterile MOPS (50 mM, pH 7.0, 10 ml) and transferred to 15-ml screw-capped bottles containing sterile MOPS (50 mM, pH 7.0, 10 ml). Aliquots (200 μl) of 14C-labelled L. monocytogenes suspension were added, and the bottles were incubated, with gentle shaking, for 24 h as described above. Glass coverslips were subsequently rinsed in sterile MOPS (50 mM, pH 7.0, 10 ml) and transferred to scintillation vials for counting by the standard method.
To obtain cell exudate, modified coryneform broth (6 ml, containing a growth-limiting concentration of glucose [1 g · liter−1]) was inoculated with 50 μl of thawed stock culture and subsequently incubated, statically, at 37°C for 24 h. The culture (6 ml) was then transferred to Eppendorf tubes and centrifuged (3 min, 13,000 rpm). Aliquots (200 μl) of the resulting supernatant were then transferred to sterile MOPS (10 ml) in 15-ml capped bottles, and glass coverslips were aseptically transferred to the bacterial exudate solution. The bottles were shaken at constant temperature for 1 h before the glass coverslips were rinsed in sterile MOPS (50 mM, pH 7.0, 10 ml) and transferred to sterile MOPS (10 ml) in 15-ml capped bottles. Radiolabelled BSA (37.5 μg) was then added, and protein adsorption was allowed to proceed for 1 h at 37°C. The coverslips were subsequently rinsed twice in MOPS (50 mM, pH 7.0, 10 ml) and transferred to scintillation vials for counting as described above.
All data from protein and cell adsorption assays were averaged over at least 10 replications, and standard deviations from the mean were calculated. The results obtained (see Fig. to ) are averages of these replications ± the standard errors.
FIG. 2 (Top) Adsorption of BSA to functionalized surfaces. (Middle) On the left, kinetics of adsorption of BSA are shown: S-NH2 (), S-C6 (○), and hydrophobized SiO2 (■). On the right, the adsorption of BSA against concentration is shown: (more ...)
Adsorption of L. monocytogenes to functionalized surfaces. The number of cells attached in the absence of BSA (open bars) and the number of cells attached to surfaces pretreated with BSA for 1 h (solid bars) are indicated.
Other analytical methods.
Fourier transform-attenuated total internal reflection infrared spectra were recorded on a Perkin-Elmer 1600 spectrometer (Perkin-Elmer, Seer Green, United Kingdom) with a SpectraTech baseline attenuated, total internal reflectance apparatus by using a 45° germanium flat-face prism purchased from Nicolet Instruments (Nicolet, Warwick, United Kingdom). Fluorescence spectra were obtained by using a Perkin-Elmer LS50B Luminescence Spectrometer equipped with a front-face sample cell. Nuclear magnetic resonance (NMR) spectra were recorded on a Jeol-EX 270 NMR spectrometer (Jeol-UK, Welwyn Garden City, United Kingdom) by using CDCl3 or CD3SOCD3 as the solvents, with residual proton signals as the internal reference. UV spectra of modified surfaces were recorded on a Perkin-Elmer Lambda 15 spectrometer.
Contact angle measurements were carried out on Krüss G-10 goniometer (Krüss GmbH, Hamburg, Germany) with a G-211 environmental cell and fitted with square pixel video capture camera; analysis of captured images was done by using Krüss Drop Shape Analysis software. Glass microscope slides derivatized as described above were used. The slides were placed in a Krüss 211 environmental chamber, and advancing and receding contact angles were measured by using three diagnostic liquids: diiodomethane, water, and ethylene glycol. Surface free energy values and the relative contributions of Lifshitz-van der Waals, electron donor, and electron acceptor components were calculated by using the method of van Oss et al. (38